The present invention refers to a method and device for the detection of microorganisms using a combination of microbiological procedures with devices constructed with fiber optics and related components.
The microbiological procedures enable the microorganisms to be selectively cultivated and these, when in physical contact with a conveniently constructed fiber optic circuit, permit the detection and monitoring of the microorganisms in a fast and accurate way.
Microorganisms are life forms that despite their micrometrical size scale significantly affect human life. They may constitute viruses, bacteria, fungi, protozoa and algae. They are present in solids, liquids and air; and in all cases it may be of vital interest or importance to detect the specific presence and/or to monitor, qualitatively or quantitatively, the growth over time of one or more of these microorganisms. Many of them are of use to various types of industries, such as food industries, brewery, viticulture, pharmaceutical, etc., for the catalysis of many biochemical reactions of commercial interest, amongst others. Other microorganisms do not seem to serve any other practical purpose, but are, in principle, harmless to human health. However, some of these microorganisms may become dangerous, for a series of reasons, after undergoing certain types of biological transmutations and whereupon they are termed pathogenic. These pathogens may exist and develop in various environments. Other types of microorganisms may be intrinsically dangerous, meaning that they are naturally constituted as noxious to human health. Overall, the presence and the concentration of some of the referred microorganisms directly affect: (i) the quality (potability and suitability for bathing)of the water for consumption in major urban centers and seaside resorts, possibly causing a series of diseases, such as diarrhea; (ii) the quality of food, which may generically cause food poisoning, and, particularly, xe2x80x9chamburger diseasexe2x80x9d and (iii) the quality of air in clinics, outpatients and hospitals where the presence of aerobiological microorganisms constitutes, for example, an important vector for hospital infections. In any of these cases, it may be of great interest or vital importance to detect the presence of certain microorganisms in real time or almost immediately, and consequently to monitor their evolution over time aiming to infer some useful information. At present, the conventional techniques for surveying microorganisms are closely linked to laboratory and outpatient procedures and based in selective biological culture and in the use of microscopy for direct visual observation. These techniques are reasonably complex, as they demand from the operator significant intervention and skill in order to obtain dependable results, and typically require 1 to 10 days for completion. This means that the conventional techniques require an average of 72 h between the capture of the microorganism, its isolation, identification and consecutive monitoring of its evolution over time. The standard procedures concerning bacteriological tests are furnished by the American Public health Association (APHA). All these procedures require incubation in a culture medium to produce an adequate supply of microorganisms for various analyses at the end of the test. Apart from which, the conventional techniques use equipment that is not particularly low cost, making its employment difficult or unviable for widespread surveying, in other words, for detection/monitoring of microorganisms in more than one place simultaneously.
There are other more modern procedures concerning biological detection, such as: radiometry, electrochemicals, chromatography, chemiluminescence, pulse field electrophoresis and fluorescence. Concerning the techniques mentioned, there are some practical restrictions such as, for example, their success is highly dependent on the quantity of bacteria that can be concentrated in the test sample. This procedure, generally, requires a minimum of 104 bacteria. In molecular reactions, the test procedures are particularly liable to cross contamination by other molecules or molecular fractions.
In addition, optical preventive techniques can be employed, aiming the elimination of pathogenic microorganisms, highlighting, for example, the use of ultraviolet light as a germicide, avoiding the infection of a determined environment or a material medium. On the other hand, the medical knowledge for fighting pathogenic microorganisms that had already infected the human body, especially those originating from hospital infection, food poisoning and water contamination, is vast but incomplete. Such microorganisms may also become resistant to any known drug due to their possible biological mutations. Recently, some success has been achieved with patients infected with these microorganisms using photochemical drugs activated by electroluminescent semiconductor diodes of high optical power with the correct wave length, permitting the total elimination of these microorganisms, without, in principle, harming the patient. This is the case, for instance, of the procedures recently developed and described by Pearce et al (H Pearce, M. Messager and J. Y. Maillard. xe2x80x9cEffect of biocides commonly used in the hospital environment on the transfer of antibiotic-resistance genes in Staphylococcus aureusxe2x80x9d. J.Hosp.
Infect. 43.2, pp. 101-107, 1999).
The optical surveying of microorganisms, which is the object of the invention herein, finds its place between the preventive techniques for the reduction/elimination of microorganisms and the techniques that aim to eliminate pathogens that have already infected the patients.
The essential characteristics of any biological sensor are its selectivity, sensitivity, resolution and response time, characterized by reactive recognition based on the type of test and the choice of detection technique. At a second level, there must be a biological surveying technology available that is robust, practical and of low cost, so that it may be employed in the field. Presently, various techniques and correlated devices are available, many of these are still at laboratory level whilst others are commercial, all aiming the detection and monitoring of microorganisms.
Microscopy, as already mentioned, constitutes a fundamental analysis tool in microbiology, not only for the detection/monitoring of microorganisms, but also for the basic study of these. In the same way, there is a method of surveying bacteria involved in processes of hospital infections presently in use, known as pulsed field electrophoresis in, which is capable of tracking with precision the microorganisms involved, mapping and evaluating the level of environmental impact. However, it presents the disadvantage of the analysis taking approximately 7 to 14 days, as described by Birron and Lai (B. Birron and E. Lai. xe2x80x9cPulsed field electrophoresis: a practical guidexe2x80x9d. Academic Press, San Diego, 1993).
With the aim of undertaking microbiological detecting and monitoring in an automatic and selective manner, the concept of the biosensor arises. A biosensor may be understood as a discreet electrical and/or optical component built based on the integration of biological materials with inorganic materials. A biosensor, in practice, is therefore capable of producing an analog electrical or optical signal when placed in contact with some specific subject, whether by its presence or any chemical-biological alterations occurring from it. A biosensor may be considered as a biologically active element, however, it requires connection, in some form, to a larger configuration, so that the electrical or optical analog can be adequately demodulated. The set-up thus formed is called a biological sensor. Fiber optics, in a general manner, may be used in the construction of biosensors, which means that through some appropriate technique, biological materials should be integrated to the sheath or core of the fiber (generally of glass or plastic) composing, therefore, an optical biosensor. In this manner, the light propagating through the core of the fiber can interact with the biological material (an antibody, for instance) integrated through a coupling by evanescent-field. The evanescent wave biosensor can induce modulation of intensity, complete phase, wave length or polarization of the propagating optical signal, or a fluorescent signal can be generated in function of a photo-biological reaction where part of the energy of this signal propagates itself through the optical fiber. Evanescent-field biosensors are described in the state of the art (J. S. Schultz. xe2x80x9cBiosensorsxe2x80x9d. Scient. Amer. pp. 64-69, 1991.; J. P. Golden, G. P. Anderson, R. A. Ogert, K. A. Breslin and F. S. Ligler. xe2x80x9cAn evanescent wave fiber optic biosensor: Challenges for real world Sensingxe2x80x9d. SPIE. 1796. 1992. pp. 1-8; S. P. J. Higson and P. M. Vadgama. xe2x80x9cDevelopment o an evanescent wave fiber optic biosensor. Med. and Biol. Eng. and Comp. 32.1994. pp. 601-609).
Biological sensors based on evanescent wave are, in a general manner, dependent on a combination of various technologies of biological, physical and chemical natures. In this sense, the North-American patents U.S. Pat. No. 4,447,546 and U.S. Pat. No. 4,558,014 describe the techniques of spectroscopy by total reflection (TRS), where the use of evanescent wave to excite a fluorescently linked analytic and, consequently, detect the resultant fluorescence is claimed. Other patents describe methods to improve measurement precision. This is the case of patent U.S. Pat. No. 5,631,170. The method involves marking the biosensor with fluorescent antibodies, in other words, antibodies linked to dye molecules. Provided that the antibodies mentioned are not linked to the antigens (subject), the dye molecules will not be excited by the evanescent-field. However, when the biosensor is in the presence of the specific antigen to be detected, the propagating light will be capable of inducing fluorescence in the dye molecules. Another method is that which is described in patent U.S. Pat. No. 4,242,447, that bases itself on the detection and quantification of bacteria in a liquid sample characterized by fact that this sample contains an agent capable of inducing the production of an enzyme in the bacteria. This enzyme shall be capable of reacting with a fluorescent conjugate ingested by the bacteria, in such a manner as to release its fluorescent part, and this total fluorescence is measured and compared to the total of bacteria present in the medium.
However, as previously mentioned, there is an urgent need for the development of a method for detecting microorganisms that is sensitive and has a quick response, without the inconvenients already mentioned, such as: necessity of a minimum of 104 bacteria, cross contamination by other molecules or molecular fractions and minimum average time of 72 h between capture of the microorganism and the monitoring of its evolution over time.
Thus, aiming to achieve this objective; a strategy of combining biological procedures with a fiber optic sensor with an evanescent-field was used, capable of detecting the microorganisms 4 to 5 times faster than when employing conventional procedures.
The aim of the present invention is the detection/monitoring of microorganisms found in air, water or foodstuffs, through the use of a fiber optic biosensor with an evanescent-field.
A first concretization of the present invention concerns a method for the detection of specific microorganisms through the application of an evanescent-field of a sensitive optical fiber, characterized by the stages:
(a) exposing the evanescent-field of the sensitive fiber optic using an appropriate technique based on physical and chemical properties;
(b) permitting immediate contact of the exposed evanescent-field obtained in the stage (a) with the sample to be examined, with the aforementioned sample having a form adequate so as to obtain the generation of an optical signal in response to the presence of microorganisms in the sample;
(c) demodulating the optical signal generated in stage (b) and using this value to quantify the microorganisms through an appropriate method.
In a second concretization, the invention is directed to a composition for use in the detection of microorganisms characterized by incorporating a selective culture medium for microorganisms needing to be detected and reactants capable of altering the properties of the medium in a manner as to favor the interaction of the system fiber-microorganism.
In a third concretization the invention refers to a device for surveying microorganisms through the insertion of a sensitive fiber optic (11), with an adequately exposed evanescent-field, into a surface or volume of a biological culture medium (12) specific for the microorganism to be detected, comprising a demodulation system based on a fiber optic circuit and related components.